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Development of an Innovative Quantification Assay Based on Aptamer Sandwich and Isothermal Dumbbell Exponential
Mathilde Aubret1,2, Maud Savonnet1,2, Patricia Laurent1
1Univ. Grenoble Alpes, CEA, LETI, Technologies for Healthcare and Biology Division, Microfluidic Systems and Bioengineering Lab, 38000 Grenoble, France.
Analytical Chemistry
|February 10, 2022
Summary
We developed a novel aptamer-based assay for sensitive and specific biomarker detection without antibodies. This antibody-free method combines aptamers with loop-mediated isothermal amplification (LAMP) for reliable disease diagnosis.
Area of Science:
- Biochemistry
- Molecular Biology
- Biotechnology
Background:
- High-sensitivity detection of blood biomarkers is crucial for early disease diagnosis.
- Aptamers offer a promising alternative to antibodies in biosensor applications, overcoming limitations of traditional methods like ELISA.
- Antibody-free assays are sought after for improved specificity and reduced cross-reactivity.
Purpose of the Study:
- To develop and validate a sensitive, antibody-free molecular quantification assay.
- To combine aptamer specificity with loop-mediated isothermal amplification (LAMP) sensitivity.
- To demonstrate the assay's potential for detecting diverse biomarkers at physiological levels.
Main Methods:
- Developed a sandwich assay utilizing aptamer-coated magnetic beads for target capture.
- Integrated a secondary aptamer into an oligonucleotide dumbbell for LAMP detection.
- Employed a two-primer LAMP strategy for exponential amplification and sensitive quantification.
- Validated the assay using an oligonucleotide mimic and the protein biomarker thrombin.
Main Results:
- Achieved a limit of detection of approximately 1 pM for oligonucleotide targets.
- Demonstrated a limit of detection of approximately 100 pM for thrombin.
- Successfully quantified low amounts of biomarkers using the aptamer-LAMP assay.
- Confirmed the assay's effectiveness in a proof-of-concept study.
Conclusions:
- The developed aptamer-LAMP assay is a sensitive and specific method for biomarker detection.
- This antibody-free approach shows significant potential for reliable disease diagnosis.
- The assay platform is adaptable for detecting various biomarkers at physiological concentrations.
